The goal of this project is to understand how hydrolysis of ATP is coupled to the gating of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) Cl channel in isolated guinea pig ventricular myocytes and in various cell lines transfected with wild-type or mutant CFTR genes. Biophysical, biochemical and molecular biological evidence indicates that the cardiac protein kinase A(PKA)-activated C1 biochemical and molecular biological evidence indicates that the cardiac protein kinase A (PKA)- activated C1 channel is close similar, if not identical, to the epithelial C1 channel, whose defective function is believed responsible for the debilitating symptoms in patients with cystic fibrosis. Results from studies of the native CFTR C1 channel in cardiac myocytes have provided a testable regulation/gating model for further structure/function investigation using electrophysiological and molecular biological techniques. Applying specific protein phosphatase inhibitors, okadaic acid and microcystin, we have identified two functionally distinct PKA phosphorylation sites in this channel molecule, one a substrate for, and the other resistant to, okadaic acid-sensitive phosphatases. Phosphorylation of the okadaic acid-sensitive site(s) activates the quiescent channel, whereas phosphorylation of the okadaic acid-insensitive site(s) enhances the channel activity by increasing the open probability. Further studies using nonhydrolyzable ATP analogs, AMP-PNP, and transition state phosphate analogs, vanadate and beryllium fluoride, suggest that hydrolysis of ATP at one nucleotide binding domain (NBD) is coupled to the opening of the phosphorylated channel, whereas binding of ATP at the other NBD increases the open probability by prolonging the lifetime of the open state conformation, and that differential phosphorylation by PKA controls the function of these two NBDs. To further understand how CFTR harvests the energy from ATP hydrolysis to fuel the conformational changes and to elaborate the structural elements in channel gating by ATP, a combination of molecular tools and several different versions of the patch-clamp technique will be applied in this project. These include site-directed mutagenesis cell-attached, excised inside-out configuration of the traditional [patch-clamping, giant excised patch methods, fast solution change and whole-cell recording with a pipette perfusion device. The questions we will address include; Is ATP hydrolysis coupled to both opening and closing of the CFTR channel? Are both NBDs functionally equivalent or complementary? What is the biochemical mechanism involved in opening of the CFTR channel? Is there a phosphorylated intermediate in the hydrolysis cycle as in the case of Na/K ATPase? Does vanadate act on NBD-1 only? Or does it, like AMP-PNP, also act on NBD-2? Where does ADP work? And what is the mechanism for ADP's inhibitor effect? How does differential phosphorylation of the R domain control gating kinetics? Which serine(s) in the R domain moderate the function of NBD-1 or NBD-2? Our understanding of the structure-function relation and the kinetic control of the rates of channel opening and closing by ATP hydrolysis should help guide the rational design of therapeutical interventions.